Recent investigations show that exogenously applied small interfering RNAs (siRNA) and long double-stranded RNA (dsRNA) precursors can be taken up and translocated in plants to induce RNA interference (RNAi) in the plant or in its fungal pathogen. The question of whether genes in the plant genome can undergo suppression as a result of exogenous RNA application on plant surface is almost unexplored. This study analyzed whether it is possible to influence transcript levels of transgenes, as more prone sequences to silencing, in Arabidopsis genome by direct exogenous application of target long dsRNAs. The data revealed that in vitro synthesized dsRNAs designed to target the gene coding regions of enhanced green fluorescent protein (EGFP) or neomycin phosphotransferase II (NPTII) suppressed their transcript levels in Arabidopsis. The fact that, simple exogenous application of polynucleotides can affect mRNA levels of plant transgenes, opens new opportunities for the development of new scientific techniques and crop improvement strategies.

Calmodulin-like proteins (CMLs) represent a large family of plant calcium sensor proteins involved in the regulation of plant responses to environmental cues and developmental processes. In the present work, we identified four alternatively spliced mRNA forms of the grapevine CML21 gene that encoded proteins with distinct N-terminal regions. We studied the transcript abundance of CML21v1, CML21v2, CML21v3, and CML21v4 in wild-growing grapevine Vitis amurensis Rupr. in response to desiccation, heat, cold, high salinity, and high mannitol stress using quantitative real-time RT-PCR. The levels of all four splice variants of VaCML21 were highly induced in response to cold stress. In addition, VaCML21v1 and VaCML21v2 forms were highly modulated by all other abiotic stress treatments. Constitutive expression of VaCML21v2 and VaCML21v4 improved biomass accumulation of V. amurensis callus cell cultures under prolonged low temperature stress. Heterologous expression of the grapevine CML21v2 and VaCML21v4 splice variants in Arabidopsis improved survival rates of the transgenic plants after freezing. The VaCML21v2 overexpression enhanced activation of the cold stress-responsive marker genes AtDREB1A and AtDREB2A, while VaCML21v4 overexpression—AtCOR47, AtRD29A, AtRD29B, and AtKIN1 genes after freezing stress in the transgenic Arabidopsis. The results indicate that the grapevine CML21 gene acts as a positive regulator in the plant response to cold stress. The detected variety of CML21 transcripts and their distinct transcriptional responses suggested that this expansion of mRNA variants could contribute to the diversity of grapevine adaptive reactions.

Plant endophytes are known to alter the profile of secondary metabolites in plant hosts. In this study, we identified the main bacterial and fungal representatives of the wild grape Vitis amurensis Rupr. microbiome and investigated a cocultivation effect of the 14 endophytes and the V. amurensis cell suspension on biomass accumulation and stilbene biosynthesis. The cocultivation of the V. amurensis cell culture with the bacteria Agrobacterium sp., Bacillus sp., and Curtobacterium sp. for 2 weeks did not significantly affect the accumulation of cell culture fresh biomass. However, it was significantly inhibited by the bacteria Erwinia sp., Pantoea sp., Pseudomonas sp., and Xanthomonas sp. and fungi Alternaria sp., Biscogniauxia sp., Cladosporium sp., Didymella sp. 2, and Fusarium sp. Cocultivation of the grapevine cell suspension with the fungi Didymella sp. 1 and Trichoderma sp. resulted in cell death. The addition of endophytic bacteria increased the total stilbene content by 2.2–5.3 times, while the addition of endophytic fungi was more effective in inducing stilbene accumulation by 2.6–16.3 times. The highest content of stilbenes in the grapevine cells cocultured with endophytic fungi was 13.63 and 13.76 mg/g of the cell dry weight (DW) after cultivation with Biscogniauxia sp. and Didymella sp. 2, respectively. The highest content of stilbenes in the grapevine cells cocultured with endophytic bacteria was 4.49 mg/g DW after cultivation with Xanthomonas sp. The increase in stilbene production was due to a significant activation of phenylalanine ammonia lyase (PAL) and stilbene synthase (STS) gene expression. We also analyzed the sensitivity of the selected endophytes to eight antibiotics, fluconazole, and trans-resveratrol. The endophytic bacteria were sensitive to gentamicin and kanamycin, while all selected fungal strains were resistant to fluconazole with the exception of Cladosporium sp. All endophytes were tolerant of trans-resveratrol. This study showed that grape endophytes stimulate the production of stilbenes in grape cell suspension, which could further contribute to the generation of a new stimulator of stilbene biosynthesis in grapevine or grape cell cultures.

Plant calmodulins (CaMs) and calmodulin-like proteins (CMLs) are important plant Ca²⁺-binding proteins that sense and decode changes in the intracellular Ca²⁺ concentration arising in response to environmental stimuli. Protein Ca²⁺ sensors are presented by complex gene families in plants and perform diverse biological functions. In this study, we cloned, sequenced, and characterized three CaM and 54 CML mRNA transcripts of Vitis amurensis Rupr., a wild-growing grapevine with a remarkable stress tolerance. Using real-time quantitative RT-PCR, we analyzed transcript abundance of the identified VaCaMs and VaCMLs in response to water deficit, high salinity, high mannitol, cold and heat stresses. Expression of VaCaMs and 32 VaCMLs actively responded to the abiotic stresses and exhibited both positive and negative regulation patterns. Other VaCML members showed slight transcriptional regulation, remained essentially unresponsive or responded only after one time interval of the treatments. The substantial alterations in the VaCaM and VaCML transcript levels revealed their involvement in the adaptation of wild-growing grapevine to environmental stresses.

Exogenous application of double-stranded RNAs (dsRNAs) and small-interfering RNAs (siRNAs) to plant surfaces has emerged as a promising method for regulation of essential genes in plant pathogens and for plant disease protection. Yet, regulation of plant endogenous genes via external RNA treatments has not been sufficiently investigated. In this study, we targeted the genes of chalcone synthase (CHS), the key enzyme in the flavonoid/anthocyanin biosynthesis pathway, and two transcriptional factors, MYBL2 and ANAC032, negatively regulating anthocyanin biosynthesis in Arabidopsis. Direct foliar application of AtCHS-specific dsRNAs and siRNAs resulted in an efficient downregulation of the AtCHS gene and suppressed anthocyanin accumulation in A. thaliana under anthocyanin biosynthesis-modulating conditions. Targeting the AtMYBL2 and AtANAC032 genes by foliar dsRNA treatments markedly reduced their mRNA levels and led to a pronounced upregulation of the AtCHS gene. The content of anthocyanins was increased after treatment with AtMYBL2-dsRNA. Laser scanning microscopy showed a passage of Cy3-labeled AtCHS-dsRNA into the A. thaliana leaf vessels, leaf parenchyma cells, and stomata, indicating the dsRNA uptake and spreading into leaf tissues and plant individual cells. Together, these data show that exogenous dsRNAs were capable of downregulating Arabidopsis genes and induced relevant biochemical changes, which may have applications in plant biotechnology and gene functional studies.

Endophytes are microorganisms that live asymptomatically inside plant tissues